Utilizing Western blots as a more sensitive assay we detected vitellogenin in queen hemolymph at the Pbm stage, approximately 60h before eclosion (Fig.2). In worker hemolymph vitellogenin was detected later at the Pha stage, about10h before eclosion (Fig. 2). It was occasionallypossible to detect vitellogenin in the worker in an earlier developmental stage (Pbd, Fig. 5A).

Juvenile hormone advances vitellogenin appearance in both castes

Exogenous juvenile hormone applied to pink- and brown-eyed pupae had a pronounced effect on vitellogenin levels in both castes, advancing the onset of hemolymph vitellogenin appearance to earlier pupal stages. Western blotanalysesindicated that in queen pupae treated with juvenile hormone, vitellogenin is expressed in PbmI, with increasing levels by Pbd, while in control pupae vitellogenin appeared only in PbmII, and remained at low levels (Fig. 3A). A similar effect was observed in worker pupae treated with juvenile hormone, which responded with an earlier appearance of vitellogenin in the Pbm II stage (Fig. 3B). Detectable vitellogenin levels were notseen in control worker pupal hemolymph during corresponding stages (Fig. 3B). Levels of total hemolymph protein concentration (Fig. 4A,B) were not affected by the juvenile hormone treatments (Mann-Whitney test >0.05).

Figure 3.

Western blot analysis to determine the onset of vitellogenin synthesis in honeybee queen and worker pupae. SDS-PAGE (7,5%) was performed with 1µg of hemolymph proteins. After blotting to a PVDF membrane vitellogenin was detected by a rabbit antiserum raised against honeybee egg proteins in combination with the PAP-DAB detection system. A) queen and B) worker pupae treated as brown-eyed pupae (Pb) with 10 µg juvenile hormone dissolved in acetone (treated), or with acetone (controls). C) Hemolymph from worker pupae of Apis mellifera treated, in Pb phase, first with a single injection of 10 µg of actinomycin D, and 1h later, with 10 µg of juvenile hormone-III. Controls received 10 µg of juvenile hormone-III. Pbl, Pbm and Pbd: brown-eyed pupae showing, light, medium, or dark pigmented cuticle, respectively. Pbm I, Pbm II and Pbm III are progressive ages within Pbm phase. Arrows indicate the 180 kDa vitellogenin band.

High levels of ecdysteroids inhibit the appearance of vitellogenin synthesis in honeybee pupal hemolymph

Injection of 5 µg of ecdysone at the Pbl stage, delayed the appearance of vitellogenin in queen and worker pupae. Ecdysone-treated worker pupae did not exhibit detectable vitellogenin levels at an advanced Pbd stage, when this protein can occasionally be detected in controls (Fig. 5A). Similarly, in ecdysone-treated queens, vitellogenin becameapparent only in the pharate adult stage (Pha), representingalmost a 48h delay as compared to its normal onset in the hemolymph (Fig. 5B). Total protein levels were significantly higher in the hemolymph of ecdysone-treated worker pupae than in control worker pupae (Mann-Whitney test <0.05; Fig. 4C).

In our experiments, exogenous ecdysteroids prevented the decay in total hemolymph protein levels that normally occurs at the end of the pupal stage. Engels (1990) observed higher protein synthesis in fat body of queens incubated in vitro in the presence of ecdysone. The declining endogenous ecdysteroid titer at the end of the pupal stage (Zufelato et al., 2000) is correlated with a decreasing protein levels in hemolymph (Michelette and Engels, 1995). These results suggest a doublemode of action of ecdysteroids on the pupal fat body. Ecdysone may prolong or enhance the expression of several pupal-specific hemolymph proteins and specificallyinhibit that of vitellogenin.

Evidence for distinct mechanisms controlling initiation and maintenance of vitellogenin synthesis in the honeybee castes

Our results indicate that the initiation of vitellogenin expression in the late pupal stages is governed by a different regulatory mechanism. Its timing correlates with an increase in late pupal juvenile hormone titer, and application of juvenile hormone III caused a caste-specific shift to earlier stages. These results may appear to contradict the earlier studies by Kaatz (1988), who carried out carefullytimed allatectomy, decapitation, and juvenile hormone rescue experiments on queen pupae. This author did not detect significant effects of allatectomy or juvenile hormone application on vitellogenin synthesis, because the incorporation rates of 3H-leucine into vitellogenin, and into hemolymph proteins in general, were very low during the late pupal stages. With a more sensitive immunological assay system we have shown that queens and workers appear to share a common control mechanism for vitellogenin induction. Juvenile hormone plays a dominant role in this mechanism, possiblyassisted by declining ecdysteroid endogenous titer. Figure 6summarizes the main results obtained on juvenile hormone and 20E- regulation of onset of vitellogenin synthesis in A. mellifera queens and workers.

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